MicroRNAs Regulating Gene Expression during Cellular Senescence and Aging

MicroRNA 在细胞衰老过程中调节基因表达

• 批准号: 7732230
• 负责人: Myriam Gorospe
• 金额: $ 26.54万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7732230
• 关键词: Affect; Aging; Aging-Related Process; Antisense RNA; Cell Aging; Cells; Collection; Diploidy; Fibroblasts; Funding; Gene Expression; Half-Life; Human; MAP Kinase Gene; MAPK8 gene; Measures; Messenger RNA; Methods; MicroRNAs; Microarray Analysis; Organism; Pattern; Phenotype; Polyribosomes; Process; Protein Overexpression; Proteins; Rate; Relative (related person); Reporter; Reverse Transcriptase Polymerase Chain Reaction; Stimulus; Stress; Translating; Translations; Tumor Suppressor Proteins; age related; insight; interest; programs; protein function; senescence; upstream kinase

During aging, organisms show altered gene expression patterns and have an increasingly impaired ability to respond to stress-causing and mitogenic stimuli. Since post-transcriptional processes critically regulate changes in the collections of expressed proteins, it is extremely important to elucidate the microRNAs (as well as RBPs, as described in other projects) that control age-related gene expression patterns. To investigate microRNA function during senescence, we employ approaches such as microRNA reduction (by transfecting an antisense RNA), microRNA overexpression (by transfecting a precursor of the microRNA), and the identification of microRNA-associated mRNAs by transfecting biotinylated microRNAs and identifying target mRNAs through various methods (eg, microarray, RT-PCR). We investigate whether microRNAs affect the stability of target mRNAs during senescence by measuring the steady-state levels and half-lives of the mRNAs of interest as a function of microRNA abundance. We investigate whether microRNAs affect the translation of target mRNAs by modulating microRNA levels, and subsequently studying the relative assocation of the mRNA with translating polysomes and by quantifying the nascent translation rates of the encoded proteins. We also employ reporter constructs to gain additional insight into the processes modulated by microRNAs and use different senescence-associated markers to examine changes in the senescence phenotype. During the past funding period, we have shown that the translation of p16 (a key senescence-associated protein that functions as tumor suppressor and potently inhibits cdk4/6) was prepressed by miR-24. miR-24 was downregulated with senescence, as were other microRNAs identified by microRNA microarray analysis. These studies also identified other microRNAs whose levels were significantly higher or lower in senescent cells than in early-passage, young cells. Ongoing studies are analyzing systematically the target mRNAs of biotinylated microRNAs whose levels decline or increase with senescence; target mRNAs are then identified using microarrays. One particular study is investigating microRNAs that control the expression of MKK4 (an upstream kinase of the MAPK JNK).

在衰老过程中，生物体显示出改变的基因表达模式，并且对应激和有丝分裂刺激的反应能力越来越受损。 由于转录后过程严格调节表达蛋白的集合的变化，因此阐明控制与年龄相关的基因表达模式的microRNA（以及RBP，如其他项目中所述）非常重要。为了研究衰老过程中的microRNA功能，我们采用了诸如降低microRNA（通过转染反义RNA），microRNA过表达（通过转染microRNA的前体）以及通过将生物素化的microRNAS和各种方法识别MICCROR（EG）通过识别MICRNAS（eg）。 我们研究了microRNA是否通过测量感兴趣的mRNA的稳态水平和半衰期作为microRNA丰度的函数来影响衰老过程中目标mRNA的稳定性。 我们研究了microRNA是否通过调节microRNA水平来影响靶mRNA的翻译，然后研究mRNA与翻译多粒子体的相对关联，并通过量化编码蛋白的新生翻译速率。 我们还采用记者构造来获得对microRNA调节过程的更多见解，并使用不同的衰老相关标记来检查衰老表型的变化。 在过去的资金期间，我们已经表明，MiR-24释放了P16的翻译（与肿瘤抑制剂一起起作用，用作肿瘤抑制和有效抑制CDK4/6的p16的翻译）。 MiR-24与MicroRNA微阵列分析鉴定的其他microRNA一样，被衰老下调。 这些研究还确定了其他microRNA的衰老细胞水平明显高或更低，而年轻细胞的水平明显高于或更低。 正在进行的研究正在系统地分析生物素化microRNA的靶标mRNA，其水平随衰老而下降或增加。 然后使用微阵列确定目标mRNA。 一项特殊的研究是研究控制MKK4（MAPK JNK上游激酶）表达的microRNA。